Abstract/Summary: For immune cells, such as macrophages, to survey tissues, mount a response against invaders, or remove dead or injured cells, they must be able to migrate. These cells are recruited to specific sites by diverse chemotactic cues, such as C-X3-C motif ligand 1 (CX3CL1) and colony-stimulating factor-1 (CSF-1). These cues signal through the CX3CL1 receptor (CX3CR1), a G-protein-coupled receptor (GPCR), and the CSF-1 receptor (CSF-1R), a receptor tyrosine kinase (RTK). Several inflammatory diseases are characterized by monocyte and macrophage recruitment by these factors. Efficient cell migration requires actin cytoskeletal remodeling, which is often regulated by Rho GTPases. The Rho GTPase family members Rac1, Rac2, and Cdc42 are thought to promote the formation of actin-rich protrusions by macrophages. However, we do not know the individual pathways by which chemoattractant receptors regulate these Rho GTPases and how they are distinct from random protrusions. To clearly define when and where these Rho GTPases are activated, we generated Frster resonance energy transfer (FRET)-based biosensors. Morphodynamic analysis of Rac1, Rac2, and Cdc42 activity in random protrusions revealed that each Rho GTPase had a distinct spatial and temporal localization pattern. Further, Rac2 appeared to play a leading role in the generation of random protrusions. Preliminary data indicate that CSF-1- and CX3CL1-stimulated protrusions had different Rac1 and Rac2 spatiotemporal activities than random protrusions. We also found that Cdc42 activity is regulated by Rac1 in response to CSF-1. These data suggest that Rho GTPases are differentially regulated depending on whether they are downstream of an RTK or a GPCR. The proposed experiments will use high-resolution imaging technologies, biosensor imaging, Rho GTPase photoactivation, and other cell biological approaches to explore the activation mechanisms of these Rho GTPase during directional cell migration. We will also explore the mechanisms by which Rho GTPases coordinate with each other to regulate chemotaxis, potentially by regulating the activity and localization of upstream and downstream signaling pathway components. Overall, our studies will provide insight into the differential regulation of Rho GTPases following signaling by RTKs and GPCRs targeting specific GEF-GTPase pathways. To date no clinically effective drugs targeting RhoGTPases are available. Therefore, the results of this study on new GEF-GTPase pathways could be exploited as potential therapeutic targets by developing specific small molecule inhibitors of GTPase activation.